Plants characterized by an increased content of methionine and related metabolites, methods of generating same and uses thereof

ABSTRACT

A method of increasing methionine and/or methionine related metabolites in a plant is provided. The method is effected by expressing within the plant a cystathionine γ-synthase encoded by a polynucleotide mutated in, or lacking, a region encoding an N-terminal portion of said cystathionine γ-synthase, said region being functional in downregulating an activity of said cystathionine γ-synthase in the plant.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to a plants characterized by an increasedcontent of methionine and methionine related metabolites, to methods ofgenerating same and to uses thereof.

The diets of humans and livestock largely consists of plant materialwhich contains low amounts of several essential amino acids notnaturally synthesized by animals or humans. As a result, the nutritionalvalue of plant material and as such plant derived foodstuff is typicallylimited, oftentimes requiring supplementation of plant derived foodstuffwith synthetic amino acids in order to increase it's nutritional value.

Efforts to improve the balance of essential amino acids in the seedproteins of important crops utilizing classical breeding and mutantselection have met with limited success on the laboratory scale andfailure on a commercial scale as agronomically acceptable cultivars havenot yet been produced.

One of the most important essential amino acid, methionine, exists inlimited quantities in legumes, cereals and other crops (Andersen J W.,“The Biochemistry of Plants”, Academic Press, NY, 1990, pp. 327-381).

The level of free methionine in plants is very low both in vegetativetissues and seeds. Methionine levels are regulated by both rate ofsynthesis and metabolism into derivative compounds. Methionine has ashort half-life due to rapid conversion to SAM and incorporation intonewly synthesized proteins. The incorporation of sulfate, methyl andcarbon into methionine and its metabolites has been analyzed in Lemnavia tracer elements. It has been observed that methionine is convertedinto SAM at a rate which is four-folds faster than the incorporation ofmethionine into proteins (Giovanelli et al., Plant Physiol. 1985,78:555).

Methionine biosynthesis is subject to regulatory control viacystathionine γ-synthase (CGS), the first enzyme in the Methioninebiosynthesis pathway (FIGS. 1 a-b). Studies conducted in Lemna haveshown that the level of CGS activity decreases in plants grown in thepresence of exogenous methionine (Thompson et al., Plant Physiol. 1982,69:1077). Conversely, treatments that induce methionine deprivationresulted in an increase in the steady-state levels of CGS (Thompson etal., Plant Physiol. 1982, 70:1347).

Thus, it was theorized that methionine may regulate its own synthesisthrough negative feedback control of cystathionine synthesis. Analysisof Arabidopsis mto1 mutants that over accumulate soluble methionine(Met) revealed that the gene encoding cystathionine gamma-synthase(CGS), the key enzyme in Met biosynthesis, is regulated at the level ofmRNA stability and that an amino acid sequence encoded by the first exonof CGS acts in cis to destabilize its own mRNA in a process that isactivated by Met or one of its metabolites. The mto1 mutations wereshown to be clustered within a small region in the exon, termed mto1,located downstream of the initiator codon (Chiba et al. Science 1999,286:1371).

Methionine synthesis is also regulated in the biosynthetic pathway ofthe aspartate family amino acids at the point of competition betweenthreonine synthase (TS) and CGS for their common substrateO-phosphohomoserine (OPH) (FIGS. 1 a-b). Evidence for TS-CGS competitionand its role in methionine synthesis has been obtained. It has beenobserved that the mto2-1 mutant of Arabidopsis over-accumulates up to20-fold more soluble methionine than the wild type plant and displays amarked reduction in threonine levels. The mto2-1 allele carries a pointmutation in the TS gene that produces a catalytically impaired enzyme(Bartlem et al. Plant Physiol. 2000, 123:101). Thus, decreased TSactivity causes methionine overproduction at the expense of threonine.Furthermore, reducing the level of CGS by antisense methods, which inturn reduce levels of methionine, leads to a seven-fold increase inthreonine levels relative to wild-type plants (Kim et al., Plant Science2000, 151:9). Therefore the levels of both TS and CGS are important inthe partitioning of OPH to Met and threonine.

Methionine deficiency in, for example, poultry diets leads to retardedgrowth, decreased feed conversion efficiency and increased fat content.As such, commercial poultry diets are typically supplemented withsynthetic Met (see, for example, U.S. Pat. No. 5,773,052).

In addition, it has been shown that wool growth in sheep and milkproduction in dairy animals are both limited by the availability of thesulfur amino acids (SAAs) methionine and cysteine (Xu et al., Dairy Sci.1998, 81:1062).

In attempts to increase the SAA content of seeds, genes encodingSAA-rich proteins from vegetative tissues have been isolated andexpressed in seeds under the control of seed-specific regulatorysequences. Such an approach has not resulted in an adequate increase intotal target amino acid content in the seed. It has been shown that thisand other approaches which attempt to increase SAA content by expressionof met-rich proteins are typically limited by the ability of plants tosynthesize methionine.

In another approach, genetic engineering methods have been utilized inan attempt to modulate the activity of enzymes catalyzing key steps ofrelevant biosynthetic pathways (see, for example, European Pat. App. No.485970).

For example, expression of a mutant form of a bacterial aspartate kinase(AK) which desensitizes negative feed-back inhibition of lysine andthreonine production in plants, resulted in a significant overproductionof threonine in vegetative tissue. However, expression of this geneunder the control of a seed-specific promoter was shown to raise thelevels of methionine content in seeds only three-fold relative to thatof wild type plants (Karchi, et al., The Plant J. 1993, 3:721).

Thus, although several approaches have been utilized in efforts toincrease the levels of methionine and other essential amino acids inplants, such approaches have failed to produce plants exhibiting asignificant increase in methionine levels.

There is thus a widely recognized need for, and it would be highlyadvantageous to have, a method of increasing the content of methionineand its related metabolites in plants, thereby increasing thenutritional and commercial value of such plants.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of increasing methionine and/or methionine related metabolites ina plant, the method comprising expressing within the plant acystathionine γ-synthase encoded by a polynucleotide mutated in, orlacking, a region encoding an N-terminal portion of the cystathionineγ-synthase, the region being functional in downregulating an activity ofthe cystathionine γ-synthase in the plant. According to yet anotheraspect of the present invention there is provided a method of increasingthe commercial and/or nutritional value of a plant comprising expressingwithin at least a portion of the plant a cystathionine γ-synthaseencoded by a polynucleotide mutated in, or lacking, a region encoding anN-terminal portion of the cystathionine γ-synthase, the region beingfunctional in downregulating an activity of the cystathionine γ-synthasein the plant, thereby increasing the level of methionine and/ormetabolites of the methionine, hence increasing the commercial and/ornutritional value of the plant.

According to still another aspect of the present invention there isprovided a method of generating a crop plant having enhanced nutritionalvalue, the method comprising: (a) obtaining a first plant expressing acystathionine γ-synthase encoded by a polynucleotide mutated in, orlacking, a region encoding an N-terminal portion of the cystathionineγ-synthase, the region being functional in downregulating an activity ofthe cystathionine γ-synthase in the plant; (b) crossing the first plantwith a second plant expressing methionine-rich storage proteins; and (c)isolating progeny plants which express the cystathionine γ-synthase andthe methionine-rich storage proteins to thereby obtain the crop planthaving enhanced nutritional value.

According to an additional aspect of the present invention there isprovided a method of generating a crop plant having enhanced nutritionalvalue, the method comprising transforming the crop plant with: (a) afirst polynucleotide encoding a cystathionine γ-synthase, the firstpolynucleotide being mutated in, or lacking, a region encoding anN-terminal portion of the cystathionine γ-synthase, the region beingfunctional in downregulating an activity of the cystathionine γ-synthasein the plant; and (b) a second polynucleotide encoding, in anexpressible form, at least one methionine-rich storage protein, tothereby obtain the crop plant having enhanced nutritional value.

According to still an additional aspect of the present invention thereis provided a method of obtaining at least one methionine relatedmetabolite or derivate, the method comprising (a) obtaining a plantexpressing a cystathionine γ-synthase encoded by a polynucleotidemutated in, or lacking, a region encoding an N-terminal portion of thecystathionine γ-synthase, the region being functional in downregulatingan activity of the cystathionine γ-synthase in the plant; and (b)extracting at least one methionine related metabolite from the plant.

According to further features in preferred embodiments of the inventiondescribed below, the plant expressing the cystathionine γ-synthase alsoover-expresses feed back insensitive aspartae kinase (AK).

According to still further features in the described preferredembodiments the plant expressing the cystathionine γ-synthase andoverexpressing feed back insensitive aspartae kinase (AK) is obtained bycrossing a first plant over expressing feed back insensitive aspartaekinase (AK) and a second plant expressing the cystathionine γ-synthaseand selecting for progeny expressing the cystathionine γ-synthase andoverexpressing feed back insensitive aspartae kinase (AK).

According to still further features in the described preferredembodiments, the methionine related metabolite or derivate is biotin.

According to still further features in the described preferredembodiments the polynucleotide is as set forth in SEQ ID NOs:3 or 4.

According to still further features in the described preferredembodiments one or more of the metabolites, such as, dimethylsulfide,contributes to the scent of a flower of a flowering plant.

According to still further features in the described preferredembodiments the plant is a consumable crop plant whereas increasing thelevel of methionine increases the level of methionine-rich storageproteins stored by the crop and thus the nutritional value of the crop.

According to yet an additional aspect of the present invention there isprovided a nucleic acid construct comprising a polynucleotide encoding acystathionine γ-synthase, the polynucleotide being mutated in, orlacking, a region encoding an N-terminal portion of the cystathionineγ-synthase, the region being functional in downregulating an activity ofthe cystathionine γ-synthase in the plant.

According to still further features in the described preferredembodiments the polynucleotide is as set forth in SEQ ID NO: 3 or 4

According to still further features in the described preferredembodiments the nucleic acid construct further comprising a promotersequence being for directing the expression of a polypeptide from thepolynucleotide sequence in a plant.

According to still further features in the described preferredembodiments the promoter is selected from the group consisting of aninducible promoter, a constitutive promoter, a tissue specific promoterand a development regulatable promoter.

According to still further features in the described preferredembodiments the nucleic acid construct further comprising an additionalpolynucleotide sequence encoding a storage protein.

According to still further features in the described preferredembodiments there is provided a plant transformed with the nucleic acidconstruct described above.

According to still further features in the described preferredembodiments the nucleic acid construct further comprising an additionalpolynucleotide encoding a transit peptide, the additional polynucleotidebeing covalently linked to the polynucleotide encoding the cystathionineγ-synthase.

According to still an additional aspect of the present invention thereis provided method of controlling ethylene levels of a plant, the methodcomprising (a) transforming the plant with a nucleic acid constructincluding a first polynucleotide region encoding a regulatable promoterbeing for directing the expression of a second polynucleotide regionencoding a cystathionine γ-synthase mutated in, or lacking, anN-terminal portion being functional in downregulating an activity of thecystathionine γ-synthase in the plant; and (b) subjecting the plant toconditions which regulate expression of the cystathionine γ-synthase,thereby controlling ethylene levels of the plant

According to still further features in the described preferredembodiments step (a) is effected at a developmental stage of the plantsuitable for altering fruit ripening.

According to still further features in the described preferredembodiments the second polynucleotide is as set forth in SEQ ID NO: 4.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing a method of generatingplants rich in methionine and/or it's metabolites.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 a is a diagram depicting biosynthetic pathways of the aspartatefamily amino acids. Major regulatory enzymes and their products areindicated. Feedback inhibition (−) and activation (+) loops are shown bydashed arrows. Abbreviations: AK=aspartate kinase;DHPS=dihydrodipicolinate synthase; HSD=homoserine dehydrogenase;HK=homoserine kinase; TS=threonine synthase; CGS=cystathione γ-synthase;CL=cystathionine γ-lyase; MS=methionine synthase; TDH=threoninedehydratase; SAM=S-adenosyl methionine; SAMS=SAM synthase.

FIG. 1 b is a diagram depicting methionine metabolism and recycling.Abbreviations: SMM=S-methylmethionine; AdoHCys=S-adenosylhomocysteine;SAM=S-adenosylmethionine; DMSP=3-dimethylsulfoniopropionate;ACC=1-aminocyclopropane-1-carboxylic acid.

FIG. 2 is a schematic representation depicting the Arabidopsis CGS cDNA.TP encodes pea rbcS-3A chloroplast transit peptide, which directs theprotein into the chloroplast and is subsequently cleaved. Nucleotidesare numbered starting at the ATG initiator codon. The primers (A1-A3)used for the PCR reactions are indicated by arrows.

FIG. 3 a is a schematic diagram depicting the DNA construct used toexpress full-length Arabidopsis CGS. Abbreviations: PRO: 35S CaMVpromoter including sequence encoding

mRNA leader; TP: pea rbcS-3A chloroplast transit peptide; 3×HA:haemaglutinin epitope tag; TER: octapine synthase 3′ terminator.

FIG. 3 b is a schematic diagram depicting the DNA construct used toexpress the 90-bp domain-deleted isoform of Arabidopsis CGS (Δ296-386).Abbreviations: PRO: 35S CaMV promoter including sequence encoding

mRNA leader; TP: pea rbcS-3A chloroplast transit peptide; 3×HA:haemaglutinin epitope tag; TER: octapine synthase 3′ terminator.

FIG. 3 c is a schematic diagram depicting the DNA construct used toexpress N-terminal domain-deleted (Δ1-519) Arabidopsis CGS.Abbreviations: PRO: 35S CaMV promoter including sequence encoding

mRNA leader; TP: pea rbcS-3A chloroplast transit peptide; 3×HA:haemaglutinin epitope tag; TER: octapine synthase 3′ terminator.

FIG. 4 is a diagram comparing the amino acid sequences of the CGSenzymes of different organisms. Boxes indicate amino acid residues thatare identical (black) or similar (gray). Dashes indicate gaps introducedto optimize the alignment. The N-terminal domain sequences of the CGSsare depicted in pink lettering. Red lettering indicates the 90-bpdeleted isoform found in the Arabidopsis cDNA (see also FIG. 3 b).Yellow lettering indicates the point mutations of the MTO1 mutant. Theblue letter in the Arabidopsis sequence at position 69 indicates thestart of the mature protein (Ravanel et al., Biochem J. 1998, 331:639).The CGS sequences compared are from Glycine max (soybean, SEQ ID NO:9),Arabidopsis thaliana (SEQ ID NO:8) Zea mays (SEQ ID NO:10), Escherichiacoli (SEQ ID NO:11) and Helicobacter pylori (SEQ ID NO: 12) GenBankaccession nos. AAD34548, U43709, AAB6 1347, P24601, AE000511,respectively. The program ClustalW was used to generate the alignment.

FIG. 5 a is a diagram depicting the nucleotide sequence of full-lengthArabidopsis CGS cDNA (SEQ ID NO: 1)

FIG. 5 b is a diagram depicting the nucleotide sequence of full-lengthArabidopsis CGS cDNA lacking sequences encoding transit peptide (used inthe construct depicted in FIG. 3 a) (SEQ ID NO: 2).

FIG. 5 c is a diagram depicting the nucleotide sequence of the 90 bpdomain-deleted Arabidopsis CGS cDNA (used in the construct depicted inFIG. 3 b) (SEQ ID NO: 3).

FIG. 5 d is a diagram depicting the nucleotide sequence of N-terminaldomain-deleted Arabidopsis CGS cDNA (used in the construct depicted inFIG. 3 c) (SEQ ID NO: 4).

FIG. 6 depicts Western blot analysis of protein from transgenic plantsexpressing Arabidopsis CGS probed with anti-3HA epitope tag antibodiesor CGS antiserum. Protein of transgenic plants expressing: N-terminaldomain-deleted CGS (truncated plants, lanes 1-3); the full-length CGS(lanes 4-6); or a 90 bp domain-deleted CGS (deleted, lanes 8-10).WT—wild-type plant. The migration of MW protein markers is indicated onthe right.

FIGS. 7 a-c illustrate levels of methionine (7 a); SMM (7 b) andmethionine incorporated into proteins (7 c) in wild-type and transgeniclines expressing full-length, truncated and deleted version ofArabidopsis CGS. The amounts of methionine and SMM were calculated fromtotal free amino acids plus SMM as detected by HPLC and is indicated asmole % of this total. The methionine level incorporated into proteinswas calculated from PBS-soluble proteins that were subjected to aminoacid analysis following protein hydrolysis by HPLC. The levels ofsoluble methionine, SMM and bound methionine were determined from leafextracts of plants grown for seven weeks. The data are presented as themean±SE of eight individual plants per line, one measurement per plant.Statistically significant differences (p<0.05) are identified byletters.

FIGS. 8 a-b illustrate patterns of volatile organic compounds (VOC)detected from wild-type plant and transgenic plants expressingfull-length, truncated and deleted version of CGS. The amount ofdimethylsulfide (8 a) and carbon disulfide (8 b) emitted from freshleaves was calculated by determined the area of the corresponding peakin the GC-MS graph as compared to a known standard. The data arepresented as the mean±SE (black bars) of eight individual plants, onemeasurement per plant.

FIG. 9 illustrates attraction of mosquitoes to plants expressing the CGSlacking its N-terminal region.

FIG. 10 illustrates ethylene production by three week old shoots oftransgenic and wild-type plants. The average±SE of five different shootsis shown.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of a method which can be used to generate isplants rich in methionine and/or methionine related metabolites and ofplants generated by this method. Specifically, the present invention canbe used to generate crop plants characterized by a higher nutritionalvalue and/or a capacity to accumulate and/or release various compoundsderived from methionine.

The principles and operation of the present invention may be betterunderstood with reference to the accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details of construction and the arrangement of the components setforth in the following descriptions or illustrated in the Examplessection. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

The production and accumulation of methionine and it's metabolites inplants is important to both plants and animals which obtain at least apart of their dietary needs from plants.

As used herein the term “plant” refers to whole plants, any plantportion, plant derived material such as mashed, chopped or otherwiseprocessed plant material and plant cells such as plant cells grown inculture.

While reducing the present invention to practice the present inventorshave uncovered that transformation of plants with a polynucleotideencoding a truncated cystathionine γ-synthase enzyme results inaccumulation of methionine and it's related metabolites in the plant.

As is further described in the Examples section which follows, theplants generated according to the teachings of the present inventionexhibit higher levels of methionine, SMM, dimethylsulfide, carbondisulfide, ethylene and biotin than wild type plants or plantsexpressing full length cystathionine γ-synthase.

Thus, according to one aspect of the present invention, there isprovided method of increasing methionine and/or methionine relatedmetabolites in a plant.

The method is effected by expressing within the plant a cystathionineγ-synthase encoded by a polynucleotide mutated in, or lacking, a regionencoding an N-terminal portion of the cystathionine γ-synthase, theregion being functional in downregulating an activity of thecystathionine γ-synthase in the plant.

Such “downregulating of activity” can result from inhibition ofcystathionine γ-synthase activity (e.g., feedback inhibition),inhibition of expression (transcription and/or translation), reducedstability of the mRNA encoding cystathionine γ-synthase, or posttranscriptional and/or post translational modifications which result indownregulation of activity. Other mechanisms are also envisaged

As is further described in the examples section which follows, such anN-terminal portion encompasses a 90 base pair region which residesdownstream of a transit peptide sequence native to plant cystathionineγ-synthase (nucleotide coordinates 296-386 of SEQ ID NO:1).

Thus, any polynucleotide sequence generated by either deleting a portionof this region, the entire region or more, as exemplified by SEQ ID NO:3 or 4, or by mutating this region in a manner which affects thesequence of the resulting transcript and optionally the sequence of thepolypeptide translated therefrom can be utilized by the presentinvention, as long as cystathionine γ-synthase activity is retained anddownregulation of this activity is at least partially abolished whenexpressed within the plant.

The polynucleotide sequence utilized by the present invention can alsoinclude a transit peptide encoding sequence for directing the formedcystathionine γ-synthase to an intracellular compartment (see theExamples section below for further detail).

Preferably, the polynucleotide sequence of the present invention forms apart of a nucleic acid construct which also includes a promoter sequencefor directing the expression of cystathionine γ-synthase in aconstitutive, tissue specific, inducible or developmentally regulatablemanner.

Numerous examples of such promoter sequences are known in the art.Examples of constitutive plant promoters include, without limitation,CaMV35S and CaMV19S promoters, FMV34S promoter, sugarcane bacilliformbadnavirus promoter, CsVMV promoter, Arabidopsis ACT2/ACT8 actinpromoter, Arabidopsis ubiquitin UBQ1 promoter, barley leaf thionin BTH6promoter, and rice actin promoter.

Particularly useful promoters for use in the present invention aretissue-specific promoters such as fruit, flower, tuber or seed specificpromoters. There are numerous examples of tissue specific promotersknown in the art. Tissue specific promoters may be used according to thepresent invention to direct methionine overproduction in tissuesconsumed as food or feed, such as seeds in cereals and tubers inpotatoes. Examples of seed-specific promoters include, but are notlimited to, the bean phaseolin storage protein promoter shown to beexpressed in a seed-specific manner in transgenic tobacco plants[Sengupta-Gopalan, 1985, Proc. Natl. Acad. Sci. USA 82: 3320-3324]; DLECand PHSβ promoters from Phaseolus [Bobb et al., 1997, Nucleic Acids Res.25: 641-7]; zein storage protein promoter [Vicente-Carbajosa et al.,1997, Proc. Natl. Acad. Sci. USA, 94: 7685-90]; conglutin gamma promoterfrom soybean [Ilgoutz et al., 1997, Plant Mol Biol 34: 613-27]; AT2S1gene promoter [Roeckel et al., 1997, Transgenic Res 6: 133-41]; ACT11actin promoter from Arabidopsis [Huang et al., 1997, Plant Mol. Biol.33: 125-39]; napA promoter from Brassica napus [Ellerstrom et al., 1996,Plant Mol Biol 32: 1019-27]. Examples of other fruit or seed specificpromoters include the E8, E4, E17 and J49 promoters from tomato [Lincolnand Fischer 1988, Mol Gen Genet 212, 71-75], as well as the 2A11promoter described in U.S. Pat. No. 4,943,674.

The inducible promoter is a promoter induced by a specific stimuli suchas stress conditions including, for example, light, temperature,chemicals, drought, high salinity, osmotic shock, oxidant conditions orby pathogenic attack. Examples of inducible promoters include, withoutbeing limited to, the light-inducible promoter derived from the pea rbcSgene, the promoter from the alfalfa rbcS gene, the promoters DRE, MYCand MYB active in drought; the promoters INT, INPS, prxEa, Ha hsp17.7G4and RD21 active in high salinity and osmotic stress, and the promotershsr203J and str246C active in pathogenic stress.

The nucleic acid construct of the present invention preferably furtherincludes additional polynucleotide regions which provide a broad hostrange prokaryote replication origin; a prokaryote selectable marker;and, for Agrobacterium transformations, T DNA sequences forAgrobacterium-mediated transfer to plant chromosomes. Where theheterologous sequence is not readily amenable to detection, theconstruct will preferably also have a selectable marker gene suitablefor determining if a plant cell has been transformed. A general reviewof suitable markers is found in Wilmink and Dons, Plant Mol. Biol.Reptr. (1993) 11:165-185.

Suitable prokaryote selectable markers include resistance towardantibiotics such as ampicillin, kanamycin or tetracycline. Other DNAsequences encoding additional functions may also be present in thenucleic acid construct, as is known in the art.

Sequences suitable for permitting integration of the polynucleotidesequence of the present invention into the plant genome are alsorecommended. These might include transposon sequences as well as Tisequences which permit random insertion of a heterologous expressioncassette into a plant genome.

Several approaches can be utilized to introduce such polynucleotidesequences into a monocotyledonous or dicotyledonous plant.

The nucleic acid construct of the present invention can be utilized tostably or transiently transform plant cells. In stable transformation,the polynucleotide of the present invention is integrated into the plantgenome and as such it represents a stable and inherited trait. Intransient transformation, the polynucleotide is expressed by the celltransformed but it is not integrated into the genome and as such itrepresents a transient trait.

There are various methods of introducing foreign genes into bothmonocotyledonous and dicotyledonous plants (Potrykus, I., Annu. Rev.Plant. Physiol., Plant. Mol. Biol. (1991) 42:205-225; Shimamoto et al.,Nature (1989) 338:274-276).

The principle methods of causing stable integration of exogenous DNAinto plant genomic DNA include two main approaches:

(i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev.Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and SomaticCell Genetics of Plants, Vol. 6, Molecular Biology of Plant NuclearGenes, eds. Schell, J., and Vasil, L. K., Academic Publishers, SanDiego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds.Kung, S. and Arntzen, C. J., Butterworth Publishers, Boston, Mass.(1989) p. 93-112.

(ii) direct DNA uptake: Paszkowski et al., in Cell Culture and SomaticCell Genetics of Plants, Vol. 6, Molecular Biology of Plant NuclearGenes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego,Calif. (1989) p. 52-68; including methods for direct uptake of DNA intoprotoplasts, Toriyama, K. et al. (1988) Bio/Technology 6:1072-1074. DNAuptake induced by brief electric shock of plant cells: Zhang et al.Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986)319:791-793. DNA injection into plant cells or tissues by particlebombardment, Klein et al. Bio/Technology (1988) 6:559-563; McCabe et al.Bio/Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990)79:206-209; by the use of micropipette systems: Neuhaus et al., Theor.Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant.(1990) 79:213-217; glass fiber or silicon carbide whisker mediatedtransformation of cell cultures, embryos or callus tissue, U.S. Pat. No.5,464,765 or by the direct incubation of DNA with germinating pollen,DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman,G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p.197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.

The Agrobacterium system includes the use of plasmid vectors thatcontain defined DNA segments that integrate into the plant genomic DNA.Methods of inoculation of the plant tissue vary depending upon the plantspecies and the Agrobacterium delivery system. A widely used approach isthe leaf disc procedure which can be performed with any tissue explantthat provides a good source for initiation of whole plantdifferentiation. Horsch et al. in Plant Molecular Biology Manual A5,Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementaryapproach employs the Agrobacterium delivery system in combination withvacuum infiltration. The Agrobacterium system is especially viable inthe creation of transgenic dicotyledenous plants.

There are various methods of direct DNA transfer into plant cells. Inelectroporation, the protoplasts are briefly exposed to a strongelectric field. In microinjection, the DNA is mechanically injecteddirectly into the cells using very small micropipettes. In microparticlebombardment, the DNA is adsorbed on microprojectiles such as magnesiumsulfate crystals or tungsten particles, and the microprojectiles arephysically accelerated into cells or plant tissues. In glass fibers orsilicon carbide whisker mediated transformation, glass fibers or siliconcarbide needles like structures are mixed with DNA and cells in asuspension to thereby induce fiber/whisker-cell collisions, which leadto cell impalement (by the fibers/whiskers) and polynucleotide injectioninto the cell.

The transformation methods described hereinabove are typically followedby propagation of transformed tissues. The most common method of plantpropagation is by seed. Regeneration by seed propagation, however, hasthe deficiency that due to heterozygosity there is a lack of uniformityin the crop, since seeds are produced by plants according to the geneticvariances governed by Mendelian rules. Basically, each seed isgenetically different and each will grow with its own specific traits.Therefore, it is preferred that the transformed plant be produced suchthat the regenerated plant has the identical traits and characteristicsof the parent transgenic plant. Therefore, it is preferred that thetransformed plant be regenerated by micropropagation which provides arapid, consistent reproduction of the transformed plants.

Micropropagation is a process of growing new generation plants from asingle piece of tissue that has been excised from a selected parentplant or cultivar. This process permits the mass reproduction of plantshaving the preferred tissue expressing the fusion protein. The newgeneration plants which are produced are genetically identical to, andhave all of the characteristics of, the original plant. Micropropagationallows mass production of quality plant material in a short period oftime and offers a rapid multiplication of selected cultivars in thepreservation of the characteristics of the original transgenic ortransformed plant. The advantages of cloning plants are the speed ofplant multiplication and the quality and uniformity of plants produced.

Micropropagation is a multi-stage procedure that requires alteration ofculture medium or growth conditions between stages. Thus, themicropropagation process involves four basic stages: Stage one, initialtissue culturing; stage two, tissue culture multiplication; stage three,differentiation and plant formation; and stage four, greenhouseculturing and hardening. During stage one, initial tissue culturing, thetissue culture is established and certified contaminant-free. Duringstage two, the initial tissue culture is multiplied until a sufficientnumber of tissue samples are produced to meet production goals. Duringstage three, the tissue samples grown in stage two are divided and growninto individual plantlets. At stage four, the transformed plantlets aretransferred to a greenhouse for hardening where the plants' tolerance tolight is gradually increased so that it can be grown in the naturalenvironment.

Although stable transformation is presently preferred, transienttransformation of, for example, flower tissue, leaf tissue, seeds,tubers or the whole plant is also envisaged by the present invention.

Transient transformation can be effected by any of the direct DNAtransfer methods described above or by viral infection using modifiedplant viruses.

Viruses that have been shown to be useful for the transformation ofplant hosts include CaMV, TMV and BV. Transformation of plants usingplant viruses is described in U.S. Pat. No. 4,855,237 (BGV), EP-A 67,553(TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809(BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications inMolecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, NewYork, pp. 172-189 (1988). Pseudovirus particles for use in expressingforeign DNA in many hosts, including plants, is described in WO87/06261.

Construction of plant RNA viruses for the introduction and expression ofnon-viral exogenous nucleic acid sequences in plants is demonstrated bythe above references as well as by Dawson, W. O. et al., Virology (1989)172:285-292; Takamatsu et al. EMBO J. (1987) 6:307-311; French et al.Science (1986) 231:1294-1297; and Takamatsu et al. FEBS Letters (1990)269:73-76.

When the virus is a DNA virus, suitable modifications can be made to thevirus itself. Alternatively, the virus can first be cloned into abacterial plasmid for ease of constructing the desired viral vector withthe foreign DNA. The virus can then be excised from the plasmid. If thevirus is a DNA virus, a bacterial origin of replication can be attachedto the viral DNA, which is then replicated by the bacteria.Transcription and translation of this DNA will produce the coat proteinwhich will encapsidate the viral DNA. If the virus is an RNA virus, thevirus is generally cloned as a cDNA and inserted into a plasmid. Theplasmid is then used to make all of the constructions. The RNA virus isthen produced by transcribing the viral sequence of the plasmid andtranslation of the viral genes to produce the coat protein(s) whichencapsidate the viral RNA.

Construction of plant RNA viruses for the introduction and expression inplants of non-viral exogenous nucleic acid sequences such as thoseincluded in the construct of the present invention is demonstrated bythe above references as well as in U.S. Pat. No. 5,316,931.

In one embodiment, a plant viral nucleic acid is provided in which thenative coat protein coding sequence has been deleted from a viralnucleic acid, a non-native plant viral coat protein coding sequence anda non-native promoter, preferably the subgenomic promoter of thenon-native coat protein coding sequence, capable of expression in theplant host, packaging of the recombinant plant viral nucleic acid, andensuring a systemic infection of the host by the recombinant plant viralnucleic acid, has been inserted.

Alternatively, the coat protein gene may be inactivated by insertion ofthe non-native nucleic acid sequence within it, such that a protein isproduced.

The recombinant plant viral nucleic acid may contain one or moreadditional non-native subgenomic promoters. Each non-native subgenomicpromoter is capable of transcribing or expressing adjacent genes ornucleic acid sequences in the plant host and incapable of recombinationwith each other and with native subgenomic promoters. Non-native(foreign) nucleic acid sequences may be inserted adjacent the nativeplant viral subgenomic promoter or the native and a non-native plantviral subgenomic promoters if more than one nucleic acid sequence isincluded. The non-native nucleic acid sequences are transcribed orexpressed in the host plant under control of the subgenomic promoter toproduce the desired products.

In a second embodiment, a recombinant plant viral nucleic acid isprovided as in the first embodiment except that the native coat proteincoding sequence is placed adjacent one of the non-native coat proteinsubgenomic promoters instead of a non-native coat protein codingsequence.

In a third embodiment, a recombinant plant viral nucleic acid isprovided in which the native coat protein gene is adjacent itssubgenomic promoter and one or more non-native subgenomic promoters havebeen inserted into the viral nucleic acid. The inserted non-nativesubgenomic promoters are capable of transcribing or expressing adjacentgenes in a plant host and are incapable of recombination with each otherand with native subgenomic promoters. Non-native nucleic acid sequencesmay be inserted adjacent the non-native subgenomic plant viral promoterssuch that said sequences are transcribed or expressed in the host plantunder control of the subgenomic promoters to produce the desiredproduct.

In a fourth embodiment, a recombinant plant viral nucleic acid isprovided as in the third embodiment except that the native coat proteincoding sequence is replaced by a non-native coat protein codingsequence.

The viral vectors are encapsidated by the coat proteins encoded by therecombinant plant viral nucleic acid to produce a recombinant plantvirus. The recombinant plant viral nucleic acid or recombinant plantvirus is used to infect appropriate host plants. The recombinant plantviral nucleic acid is capable of replication in the host, systemicspread in the host, and transcription or expression of foreign gene(s)(isolated nucleic acid) in the host to produce the desired protein.

As is described in detail in the Examples section which follows, theplants generated according to the teachings of the present invention arecharacterized by several commercially important traits.

It has been shown that the protein storage capacity of numerous cropplants is limited by a level of methionine found in these plants. Thus,the ability of plants generated according to the teachings of thepresent invention to accumulate higher levels of methionine enables suchplants to generate and accumulate higher levels of proteins thus greatlyincreasing the nutritional and commercial value of such plants.

To further increase the level of plant expressed proteins, such as, forexample, storage proteins (see examples below), transformed plantsexpressing the truncated or mutated cystathionine γ-synthase can becrossed with plants expressing high levels of a storage protein and aprogeny expressing both can be selected.

Alternatively, plants can be co-transformed with a first polynucleotideencoding the truncated or mutated cystathionine γ-synthase of thepresent invention and a second polynucleotide encoding a storage proteinsuch as, but not limited to, a maize 15 kDa zein protein which contains12% methionine by weight; a maize 10 kDa zein protein which contains 22%methionine by weight; a Brazil nut seed 2S albumin which contains 24%methionine by weight; a sun flower seed methionine-rich protein, and arice 10 kDa seed prolamin which contains 25% methionine by weight.

Co-transfection of plants with the first and second polynucleotides canbe effected using two nucleic acid constructs each including a specificpolynucleotide or alternatively both polynucleotides can be introducedinto the plant via a single nucleic acid construct.

Several approaches can be utilized to enable expression of bothpolynucleotides from a single construct.

The expression of each polynucleotide can be directed by a dedicatedpromoter or alternatively, the first and second polynucleotide sequencescan be transcribed from a single promoter as a polycistronic message. Insuch a case, the polycistronic sequence also includes an internalribosome entry site (IRES) to facilitate translation of the downstreampolynucleotide sequence.

Still alternatively, the first and second polynucleotide sequences canbe translationally fused around a protease cleavage site cleavable by aplant expressed protease, in which case, protease cleavage of thechimeric polypeptide formed would generate the cystathionine γ-synthaseand storage protein.

It will be appreciated that expression of mutant or wild-type aspartatekinase (AK) in any of the plants described above can further increasemethionine production and accumulation/assimilation and/or storageprotein content in such plants.

Expression of aspartate kinase (AK) (preferably a feed back insensitivemutant form) can be achieved by either directly transforming the plantsdescribed above with a polynucleotide sequence encoding such an AK, oralternatively by crossing any of the plants described above with asecond plant expressing (preferably high levels—over expression) the AK.

Expression of AK would shift the carbon skeleton towards the threoninebranch of the aspartate family, and would thus lead to an increase ofthreonine and methionine synthesis.

In any case, the resulting plants would be able to generate andaccumulate high levels of a storage protein or proteins thus greatlyincreasing the commercial and/or nutritional value of such plants.

It will be appreciated that since such plants can provide a rich sourceof protein to both animals and humans, they are suitable for cultivationin geographical regions in which both animals and humans suffer frommalnutrition, such as the case with third world countries.

The plants of the present invention are also characterized by highlevels of methionine related metabolites/derivates such as SMM,dimethylsulfide, carbon disulfide, ethylene and biotin. Such metabolitesmay be extracted from the plants using methods known to the schooledartisan, such as, but not limited to, solvent extraction, filtration,chromatography, electrophoresis and the like.

As is further described in the Examples section which follows, some orall of these metabolites participate in plant pathogen resistance,allelopathy or scent production.

As such, the plants generated according to the teachings of the presentinvention, would exhibit increased pathogen resistance, allelopathiccapabilities which are important in limiting growth conditions, anincreased or altered scent which can lead to an increase in pollinationby insects or other vectors and/or an increase in the commercial and/ornutritional value of the plant.

Since expression of the modified cystathionine γ-synthase of the presentinvention in plants has led to increased levels of ethylene, the nucleicacid constructs of the present invention can also be used to selectivelyincrease ethylene levels in plants for the purposes of, for example,controlling fruit ripening or plant development.

Thus according to another aspect of the present invention there isprovided a method of controlling ethylene levels of a plant. The methodaccording to this aspect of the present invention is effected bytransforming the plant with a nucleic acid construct including a firstpolynucleotide region encoding a regulatable promoter (e.g., inducible,tissue specific, developmentally regulated) which serves for directingthe expression of a second polynucleotide region encoding acystathionine γ-synthase mutated in, or lacking, an N-terminal portionbeing functional in downregulating an activity of the cystathionineγ-synthase in the plant (preferably that encoded by SEQ ID NO:4).Following transformation and generation of seedlings, positivetransformants are selected by standard detection methods (e.g., PCR) orby subjecting the seedlings to conditions suitable for inducingexpression of the cystathionine γ-synthase and comparing ethylene levelsof transgenic seedlings to that of wild type seedlings.

Once a line of transformants is established, the expression of thecystathionine γ-synthase is regulated by either switching expression onand off using inductive conditions (described hereinabove), or the plantis allowed to grow to a desired stage in which expression of theexogenous cystathionine γ-synthase is switched on and ethyleneproduction is increased.

In any case, the above describe methodology can be used to control fruitripening or plant development, capabilities which are of high commercialimportance.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Materials and Methods

Vector construction: Arabidopsis CGS sequences were cloned as follows.Briefly, total RNA from flowers and leaves was extracted withTri-Reagent (Sigma), according to the protocol provided by themanufacturer. Complementary DNA was synthesized from 1 μg total RNA byAMV reverse transcriptase (Promega, Madison, Wis.) and CGS cDNAs werecloned by PCR using primers listed in Table 1. cDNAs encoding thefull-length and the 90-bp deleted CGS (FIG. 2) were amplified, usingprimers A1 (SEQ ID NO: 5) and A2 (SEQ ID NO: 6) and primers A3 (SEQ IDNO: 7) and A2 (SEQ ID NO: 6), respectively.

TABLE 1 PCR primers Starting nucleotide coordinate (bp's 3′ of CGS cDNAPrimer ATG initiator codon) Nucleotide sequence A1 2045′ AGCATGCTCGTCCGTCAGC TGAGCATTAAAGC 3′ (SEQ ID NO: 5) A2 16925′ ACCCGGGGATGGGCTTCGA GAGCTTGAAG 3′ (SEQ ID NO: 6) A3 5195′ AGCATGCTCAGCTCCGATG GGAGCCTCACTG 3′ (SEQ ID NO: 7)

Primers A1 and A3 encode an SphI restriction site containing an ATGtranslation-initiation codon, while primer A2 contains a SmaIrestriction site. PCR and reverse transcription (RT)-PCR analyses wereperformed in transfection experiments according to established protocols(Frohman, M. A., “PCR Protocols: A Guide to Methods and Applications”,1990, Academic Press, San Diego). Amplified CGS sequences were clonedinto vector pGMT (Promega) and sequenced with an automatic DNA sequencer(Model 373A, Applied Biosystems, Foster City, Calif.).

Polymerase chain reactions were performed using Expend DNA polymerase(Boehringer) and consisted of 30 cycles of the following set ofincubations: 1 min./95° C.→1 min./54° C.→1.5 min./72° C. PCR productswere ligated into plasmid pGMT and both DNA strands of inserts weresequenced to control for fidelity of amplification. Following digestionwith SphI, the PCR-derived inserts were subcloned into SphI-linearizedCE vector (Shaul et al., Plant Physiol. 1992, 100:1157; Karchi et al.,The Plant J. 1993, 3:721). Vector CE expresses genes in vegetativetissues and targets synthesized protein to plastids. It contains the 35Spromoter of cauliflower mosaic virus and the

-DNA sequence of the coat protein gene of tobacco mosaic virus whichencodes a translation-enhancing mRNA leader (Gallie et al., Plant Cell1989, 1:301). Targeting of expressed protein to plastids is directed viapea rbcS-3A (rubisco) transit peptide (TP) encoded by a sequenceinserted between the

-DNA and CGS coding sequences. The SmaI-digested fragment of vector CEcontaining the 35S promoter and the rubisco TP and CGS coding sequenceswas subcloned into SmaI-digested binary Ti plasmid, pZP111 (Hadjukiewiczet al., Plant Mol. Biol. 1994, 25:989), thus generatinginsert-containing pZP111 constructs. Tang et al. [Tang at al. 2000,Plant J 23:195-203] designed the pZP111 plasmid with SmaI site at theend of its polylinker site, and an epitop tag of a triple haemaglutininepitope-tag (3×HA), followed by a TGA stop codon and a 3′ terminatorderived from the octopine synthetase gene of Agrobacterium tumefaciens.The SmaI site enables in-frame fusions with the CGS sequences at the 3′end of the insert. Plasmid pZP111 a also provides a gene for kanamycinresistance.

Plant transformation: Insert-containing pZP111 constructs were used totransform Nicotiana tabacum cv Samson NN by the leaf-disk protocol(Hosch et al., Science 1985, 227:1229). Transgenic plants were selectedon the basis of their ability to regenerate and root on media containing100 mg/l kanamycin. They were further grown on Nitsch medium in magentaboxes at 25° C., with a light intensity of 30-40 mE m⁻² sec⁻¹ and a 16/8h day/night regime. Approximately month-old leaves were tested by PCRfor the presence of the CGS gene. Thirty transgenic plants from each setof transformations were then transferred to the greenhouse, grown tomaturity and selfed.

Western immunoblotting analysis of transgenic protein expression: Leaveswere homogenized by mortar and pestle in an equal volume of cold 100 mMTris-HCl pH 7.5 containing 2 mM EDTA and 1 mMphenylmethylsulfonylfluoride (PMSF). Homogenates were centrifuged (5min. at 16000 g at 4° C.) and supernatants were harvested. Supernatantproteins were fractionated by SDS-PAGE, transfer-blotted to a PVDFmembrane using a Bio-Rad Protein Trans-Blot apparatus and blots wereblocked by overnight incubation at 4° C. in a solution containing 5%non-fat milk. Blots were incubated with anti-3HA antibodies or CGSanti-serum for 2 h at room temperature and subsequently incubated withhorseradish-peroxidase-conjugated anti-mouse IgG for 2 h at roomtemperature. Visualization of labeled proteins was performed via anenhanced chemiluminscence immunodetection kit (Pierce), in accordancewith the manufacturer's instructions.

Measurement of free amino acid levels in leaves and seeds of transgenicplants: Leaves from approximately month-old, greenhouse-grown tobaccoplants were harvested, ground in liquid nitrogen and stored frozen. Freeamino acids were extracted from ground and frozen leaves as previouslydescribed (Bieleski et al., Anal Biochem. 1966, 17:278). Concentrationsof free amino acids were determined using O-phthalaldehyde reagentfollowed by measurement of 335/447 nm fluorescence. Amino acidcomposition was determined in samples containing 66 nmol of total freeamino acids using a Hewlett-Packard Amino Quant Liquid Chromatograph.

Measurement of protein-bound amino acids levels: Leaves were homogenizedand extracted at 4° C. in 100 mM Tris-HCl, pH 7.5 containing 1 mM PMSFand 1 μM leupeptin. Following centrifugation (5 min, 16,000 g, 4° C.) ofhomogenates, supernatants were collected and protein concentration wasdetermined by the Bradford method. Protein (30 mg) was hydrolyzed in 0.3ml distilled 6 N HCl at 110° C. for 22 h, under vacuum. The compositionof protein-bound amino acids was identified by loading 4 μg ofhydrolyzed protein on an HPLC column (Dionex, Bio LC Amino AcidAnalyzer).

Gas chromatography and mass spectrometry: The volatile organic compounds(VOCs) from leaves of transgenic plants were determined, using the OI4560 Purge and Trap system connected to a Hewlett Packard 5890 series IIGas Chromatograph equipped with a 5972 MS Detector. Data analysis wasperformed using HP MS Chemstation software according to EPA method No524.2 using 60 m fused silica capillary columns (ID of 0.25 μm and filmthickness of 1.4 μm). Double-distilled water (10 ml) was added to 1 g offresh leaves taken from young transgenic plants, samples were purged atroom temperature with 99.999% helium (40 ml per min for 11 min) and VOCswere isolated in an ambient temperature micro-trap containing silica,Tenax and charcoal as adsorbents. Volatile analytes were then thermallydesorbed at 180° C. and injected into the gas chromatographic columnthrough a transfer line heated to 100° C. Chromatographic separation wasperformed over a temperature gradient rising from 35° C. to 220° C. at arate of 10° C. per minute.

Ethylene production assay: Ethylene production was assayed as previouslydescribed (Guzman and Ecker, Plant Cell 1990, 2:513) from young(approximately 10 cm high) regenerated transgenic shoots. Shoots wereplanted in soil, allowed to grow for three weeks and, were incubatedwith their pots for 24 h at 22° C. in airtight 1 L magenta. Ethylene wasassayed by GC (HP, model 5890) using an Alumina 60/80 column (ModelSupleco 020283). An appropriately diluted standard of 640 μl L⁻¹ ofethylene (balance N₂) was used to calculate ethylene concentration.

Measurement of biotin: Leaf samples were frozen in liquid nitrogen andbiotin levels were measured as previously described (Chang et al., J.Biochem. Biophys. Methods 1994, 29:321) using alkalinephosphatase-conjugated avidin (Extravidin, Sigma) instead ofstreptavidin-HRP.

Experimental Results

The catalytic domain of CGS is located in the C-terminal domain:Alignment of bacterial and plant CGS sequences reveals that theN-terminal domain of the plant CGS, where the mto1 mutations arelocated, does not exist in bacterial CGS (FIG. 4). In order to study therole of this domain in Arabidopsis thaliana CGS (AtCGS) activity, AtCGSsequences, full-length or containing a deletion of this domain, werecloned into prokaryotic vector pQE (QIAGEN) which was used to transformthe CGS-deficient E. coli mutant LE392 (metB). As a positive control,the entire open reading frame of E. coli CGS was also transformed in themutant. All of these constructs allowed the mutant to grow on minimalmedium lacking Met, whereas vector-only control transformants could not.Therefore, it can be concluded that N-terminal domain-deleted CGS isable to functionally complement the E. coli mutant and hence that thecatalytic domain of CGS is located in the conserved C-terminal domain ofthis enzyme. We further suggest that the N-terminal domain of CGS servesan additional plant-specific regulatory role in the function of CGSs.

An alternate transcript of CGS containing a 90 bp in-frame deletionwithin the N-terminal domain: CGS-encoding DNA was PCR-amplified from anArabidopsis flower cDNA library using primers A1 and A2 (FIG. 2, Table1). Two amplification products were generated; one with a sizecorresponding to full-length CGS and the second shorter by about 0.1 kb.The sequence of the longer product was found to correspond to thatexpected for full-length CGS (Genbank accession number U43709) while thesmaller one was found to contain a 90 bp deletion within the N-terminaldomain, between nucleotides 296 and 386 (FIG. 2 and at protein level seeFIG. 4, red letters). This 90 bp deletion was found to keep the proteinsequence in frame, suggesting that protein functionality, includingcatalytic activity, was retained on the C-terminal side of the deletion.The sequences of the full-length CGS and that of its deleted version areshown in FIG. 5.

Primers A1 and A2 were also employed to amplify CGS cDNA from anArabidopsis hypocotyl cDNA library and, via RT-PCR, from total RNAextracted from Arabidopsis leaves, flowers, roots and seedlings. In allcases, both the full-length and the 90 bp domain-deleted cDNA sequenceswere amplified. Sequence analysis of the Arabidopsis database failed toidentify a separate CGS gene lacking this 90 bp domain, implying thatboth the full-length and the deletion-containing cDNAs are produced fromthe same gene. The 90 bp deletion is contained within exon 1 of the CGStranscript (FIG. 2) and is not flanked by any known consensusintron/exon boundary sequences. Its vicinity to the MTO1 region 45 basesdownstream (FIG. 2), which is important for post-transcriptionalregulation of CGS levels, suggests that this deleted region may beinvolved in regulating CGS expression.

Generation of tobacco plants transgenic for deletion mutants of CGS: Inorder to analyze the role of the deleted 90 bp domain and of theN-terminal domain of CGS in plants, constructs encoding full-length, 90bp domain-deleted (Δ296-386) and N-terminal domain-deleted (Δ1-519)Arabidopsis CGS cDNA were prepared (FIGS. 3 a, 3 b and 3 c,respectively). Since the N-terminal domain-deleted construct does notpossess a transit peptide, and to shorten the sequence, the original TPin these constructs has been removed and replaced by pea rbcS-3A TPfused in-frame to the CGS coding sequence. These constructs furthercomprise an epitope tag (3HA) at their 3′ termini, enabling detection ofexpressed protein by Western blot, distinction of transgenic fromendogenous protein and establishment of whether a single DNA or cDNAsequence can give to both the full-length and the 90 bp-deleted isoformsof CGS. Transgenic tobacco plants were generated using these threeconstructs.

Analysis of tobacco plants transgenic for N-terminal domain-deleted and90-bp domain-deleted CGS: Transgenic T0 tobacco plants were selected, onsterile media containing 100 mg/l kanamycin sulfate. Thirtykanamycin-resistant plants from each transgenic line were analyzed byPCR for the presence of the CGS transgenes. All plants were found tocontain their respective transgenes. Expression of transgenic CGSprotein in vegetative tissues of T0 plants was analyzed via Westernimmunoblotting with anti-3HA antibodies or CGS anti-serum. FIG. 6 showthe results of several representative plants expressing the various CGStransgenes. Protein species corresponding in size to the expected formsof CGS were expressed at varying levels, indicating that CGS isprocessed as it is transported into plastids. In plants expressing highlevels of full-length CGS protein, an additional species of a sizecorresponding to that of the 90 bp-deleted protein was detected,suggesting that both isoforms are transcribed from a single gene.

Phenotype of CGS-transgenic tobacco plants: The phenotype of sevenweek-old, kanamycin-resistant, greenhouse-grown plants was assessed.Plants transgenic for full-length CGS generally displayed a phenotypesimilar to wild-type, however plants transgenic for N-terminal-deletedCGS exhibited stunted growth and delayed development, and had narrower,greener and curlier leaves. These characteristics were most noticeableat later stages of plant development. Furthermore, the plants lost theirapical dominance, buds formed on primary stems, and displayed bushystructures. Buds were produced in the upper stems but most were cut offfollowing formation of an abscission zone and some, mostly sterile,flowers formed. The life-span of these plants was found to be 2.5 years,a dramatic extension from their normal lifespan of 3-6 months. Theseplants were perhaps enabled to survive due to their sustained growth ofsecondary stems.

Most of the transgenic plants expressing the 90 bp-deleted CGS geneexhibited a similar phenotype to those expressing full-length CGS andthe wild type plants. However, some of the plants expressing high levelsof the 90 bp-deleted transgenic protein were slightly abnormal,exhibiting greener and narrower leaves than the wild-type. Thisphenotype was diminished in latter developmental stages.

Emmission of dimethyl sulfide from transgenic plants expressingN-terminal domain-deleted CGS: One of the most notable characteristicsof transgenic plants expressing the N-terminal-deleted CGS is a typical,very heavy odor. In order to determine the identity of the chemicalcausing this odor, GC-MS was analysis was performed on 1 g of youngleaves as described in the methods section. As shown in FIGS. 8 a-b,transgenic plants were found to principally emit the VOC dimethylsulfideand carbon disulfide, both catabolic products of Met. In one transgenicplant (N60), emission of dimethylsulfide was found to be 60-fold higherthan that in wild-type plants. Although broad variation indimethylsulfide production was observed overall within the transgenicplants, those expressing N-terminal-deleted CGS were found to produce 21times the levels of dimethylsulfide as compared to wild-type plants ortransgenic plants expressing the full-length CGS (FIGS. 8 a-b). In mostcases a positive correlation was observed between expression levels ofCGS, as indicated by Western immunoblot analysis, and dimethylsulfideproduction levels. In addition, changes in the levels of other compoundssuch as pentanal, hexanal and hexanol were also observed (Table 2).

The heavy odor emitted from plants expressing the CGS lacking itsN-terminal region attracted small mosquitoes as shown in FIG. 9.

TABLE 2 Analysis of transgenic plants expressing the Arabidopsis CGScDNA SMM Met Quantity (GCMS results × 103 units) Plant western (% free(% free Dimethyl Carbon ID # phenotype blot amino acid) amino acid)sulfide disulfide Pentanal Hexanal Hexanol N5 − + 0 0 60 110 8 95 15N11 + +++ 6.2 2.9 680 0 0 100 0 N23 + ++ 7.2 0.9 230 35 6 12 8 N25 + +++10.5 1.7 400 190 6 18 11 N37 + +++ 6.9 0.9 250 0 30 390 200 N44 + ++ 6.71.0 530 550 0 48 0 N46 − ++ 15.9 11.2 980 0 10 8 0 N63 + +++ 8 0.92 450220 0 280 144 N66 + +++ 10.4 2.7 1250 0 13 50 35 D40 + +++ 13.8 1.8 26090 10 92 30 D44 − +++ 16.2 1.5 240 0 10 38 0 D48 − + 5.7 0.7 160 22 0 6228 D53 − +++ 13.0 7.2 500 38 0 58 36 D55 − ++ 12.4 0.5 500 60 8 110 20D56 − ++ 9.7 2.7 48 12 16 100 32 D67 − +++ 7.8 5.0 140 0 8 32 0 D70 + ++10.5 2.5 132 7 17 27 0 D71 + ++ 9.9 1.1 240 80 20 134 35 D76 − ++ 8.14.1 190 18 8 96 0 F23 − +++ 4.9 0.3 24 5 8 50 0 F30 − +++ 5.4 1.1 130 0350 130 0 F39 − +++ 6.4 0.7 310 59 0 200 32 F41 − ++ 10.8 1.9 7 0 34 750 F45 − + 12.5 0.6 40 11 0 200 0 F47 − ++ 10.2 1.0 9 0 13 131 21 F63 −++ 3.0 0.2 60 0 17 110 0 F66 − ++ 8.9 0.5 5.5 0 5 27 0 F71 − +++ 4.6 3.417 0 3.5 23 8.5 NN10 − − 0.8 0 15 5 5 0 22 NN11 − − 1.6 0 26 16 8 58 68NN12 − − 0.5 0.2 5 55 6 80 0 Transgenic lines denoted by N# expressN-terminal domain-deleted CGS and those denoted by D# express 90 bpdomain-deleted CGS. Transgenic lines expressing full-length CGS aredenoted by F#. Wild-type plants are denoted by NN#. Levels of Met andSMM are calculated as % mole from total amino acids. Results from GC-MSanalysis are also indicated.

Amino acid analysis of transgenic plants: Free amino acids wereextracted from young leaves and levels of Met and SMM, as % total freeamino acids, were determined. Levels of Met were found to be higher inall three transgenic lines expressing CGS at moderate to high levelsthan in the wild-type plants (FIGS. 7 a,b). The highest levels observed(11.2%, plant N46) represent a 56-fold increase relative to wild-type.Average Met levels were found, in order of decreasing percentage, to be2.7% in plants expressing 90 bp-deleted CGS, 1.7% in plants expressingN-Terminal-deleted CGS, 1.1% in plants expressing full-length CGS. and0.1% in wild-type plants. The same general trend was detected in theaverage SMM levels (FIG. 8 b); Overall, levels of SMM in transgenicplants were increased 7- to 16-fold over wild-type plants.

Levels of protein-incorporated Met were also analyzed in leaves oftransgenic plants (FIG. 7 c). Levels of protein-incorporated Metincreased 2-fold in plants expressing N-terminal domain-deleted CGSrelative to wild-type. Thus, overexpression of CGS, obtained viadeletion of negative regulatory sequences in exon 1, leads to increasedlevels of Met and SMM production in the transgenic tobacco plants.Importantly, the levels of the other aspartate family amino acids,threonine, lysine and isoleucine, remained unchanged in these plants.

Ethylene production: Ethylene, one of the major phytohormones in plants,is synthesized from methionine via SAM. Since the transgenic plantsexpressing the truncated CGS possesed a phenotype that resemblesethylene symptoms, the rate of ethylene emission in three-week-oldshoots regenerated from transgenic and wild-type plants was tested. Asshown in FIG. 10, ethylene production was comparable between wild-typeand transgenic plants expressing full-length and deleted version ofArabidopsis CGS. However, in the transgenic plants expressing thetuncated CGS which lacks the N-terminal region, ethylene production wasnearly 40 times higher than that of wild-type plants. This high level ofethylene may explain some of the abnormal phenotypes observed in thetransgenic plants, such as enhanced abscission of flower buds, drynessof the apical meristem and leaf primordium, retarded stem elongation, aswell as curled leaves.

Biotin production: As shown in Table 3, biotin levels in the transgenicplants expressing the N-terminal domain deleted CGS were found to be 20%higher than that of wild-type plants.

TABLE 3 Biotin levels in plants expressing N-terminal domain-deleted CGSversus that in wild-type plants (NN) Picogram biotin/ Picogram biotin/Strains μg tissue Wild-type μg tissue N13 9.45 NN1 10.18 N14 9.48 NN28.32 N44 9.04 NN3 5.35 N46 16.29 NN4 8.03 N63 10.23 NN5 10.02 Average10.9 Average 8.5 S.D 1.37 S.D 0.77

Discussion

The results presented by this study bring forth evidence that CGSactivity is the major rate-limiting step in methionine biosynthesis. Inaddition, several new findings concerning the regulation of methioninebiosynthesis and methionine accumulation and catabolism have also beenuncovered by this study.

As shown herein, the N-terminal of CGS, a region not essential forenzymatic activity is important in its regulation. Such regulation mayoccur at the level of transcript stability, as suggested by Chiba et al.[Chiba et al. (1999) Science 286: 1371-1374], who demonstrated thatmutations clustered in small part at this N-terminal region can lead tomRNA stability and thus enhance enzyme activity, or alternatively suchregulation can occur at the post translational level.

As described hereinabove, a marked increase of methionine levels wasobserved in the transgenic tobacco plants overexpressing the truncatedCGS. However, methionine related metabolite levels also increased intransgenic plants overexpressing the truncated CGS, implying thatmethionine levels can not be raised beyond a certain threshold intobacco plants.

In addition, the transgenic plants of the present invention exhibited atwo fold increase in protein-bound methionine. Analysis of thecharacteristic smell of the transgenic plants overexpressing thetruncated CGS revealed that methyl and sulfide groups are emitted fromthe plants. The fact that dimethylsulfide and carbon disulfide are themajor compounds emanating from the plants, at a level more than 60 timesthat of the wild type plants, suggests that the methionine level is notregulated by the sulfur supply or by the cysteine level.

Although plant CGS lacking an N-terminal is similar to bacterial CGS,overexpression of the bacterial gene would not produce similar resultssince bacterial CGS enzyme utilizes O-succinylhomoserien rather thanO-phosphohomoserine as a substrate in the methionine pathway.

Sulfur Emission:

As mentioned hereinabove, analysis of the characteristic smell of thetransgenic plants overexpressing the truncated CGS revealed that methyland sulfide groups are emitted from the plants.

Several possible functions have been attributed to sulfur emission byplants, including its anti-microbial effect, the discharge of toxiccompounds produced in cellular metabolism, or allelopathic effects inplant communities. Dimethylsulfide and methanthiol are a precursors forvarieties of phytoalexins that are involved in the defense responses.This compound is induced by attacks by herbivores (including insects),and deters them [Stadler (2000) in: Sulfur Nutrition and Sulfurassimilation in Higher Plants, Paul Haupt, Bern, Switzerland pp.187-202; Attieh et al. (1995) J. Biol. Chem. 270: 9250-9257].

In addition, it has been suggested that dimethylsulfide contributes tothe scent of flowers, and is an important element in the flavor of manyvegetables, beer and tea [Mandin et al. (1999) J. Agric Food Chem47:2355-2359].

Production of SMM:

Analysis of free amino acids extracted from the transgenic tobaccoplants revealed a level of S-methylmethionine (SMM) about ten timeshigher than that found in wild type plants.

SMM has several important functions in plants. It is produced in allflowering plants from methionine, which has a separate mechanism toconvert SMM back to methionine. It functions in both storage andtransport of labile methyl moieties. It is thought to be the major formof reduced sulfur capable of phloem movement.

In barley for example, SMM stores methyl groups which are converted backto methionine in periods of high metabolism, such as in the early stagesof germination [Pimenta et al. (1998) Plant Physiol. 118:431-438]. Inwheat, it has been suggested that SMM which is synthesized in leavestranslocates to the grains and is recycled to methionine for use inprotein synthesis.

SMM is also a precursor for 3-dimethylsulfoniopropionate (DMSP), anosmoprotectant accumulated by certain plants. Salinization generallyenhances DMSP accumulation which is an analog of a betaine having asulfur atom in place of nitrogen and exhibiting osmoprotectant andcryoprotectant properties similar to that of betaines. When nitrogensupply is limited, higher plants and algae may compensate for reducedcapacity to synthesize betaines by producing more DMSP [James et al.(1995) Plant Physiol. 108:1439-1448].

Production of SAM:

The level of S-adenosyl methionine (SAM) was not measured in this study,since it is a very unstable compound and as such difficult to measure.however, an increase in the level of SAM products, biotin and ethylene,in the transgenic tobacco plants indicates higher SAM production in thetransgenic plants.

SAM serves as a cofactor for a variety of reactions in all living cells.It is the major methyl-group donor in numerous highly specifictransmethylation reactions of proteins, lipids, polysaccharides andnucleic acids. These reactions may also play a crucial role inredirecting intermediates toward specific biosynthetic pathways[Belbahri et al. (2000) Biotechnol. Bioeng. 69:11-20]. In tobacco cells,for example, putricine-N-methyltransferase catalyzes the transfer of amethyl group from SAM to an amino-group of puticine, which is the firststep in the biosynthesis of tobacco alkaloids. The lack of SAM mayaffect this metabolic pathway by altering the product quantitatively andqualitatively. Attempts have been made to increase alkaloid contents intobacco (nicotine and nornicotine), through transgenic cellsoverexpressing SAM-synthase 1.

Thus, the present invention also provides an alternative approach toincreasing SAM and metabolites such as nicotine and nornicotine inplants.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications patents, patent applicationsand sequences identified by their accession numbers mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent, patent application or sequence identified by theiraccession number was specifically and individually indicated to beincorporated herein by reference. In addition, citation oridentification of any reference in this application shall not beconstrued as an admission that such reference is available as prior artto the present invention.

1. A method of increasing methionine and/or methionine relatedmetabolites in a plant, the method comprising expressing within theplant an exogenous polypeptide having a cystathionine γ-synthasecatalytic activity, said polypeptide having an amino acid sequencecorresponding to SEQ ID NO: 8 and further having at least a mutationand/or deletion in a region corresponding to amino acid coordinates 99to 131 of SEQ ID NO: 8, thereby increasing methionine and/or methioninerelated metabolites selected from the group consisting of S-methylmethionine, ethylene, dimethylsulfide, carbon disulfide or biotin insaid plant as compared to methionine and/or said methionine relatedmetabolites in a similar wild type plant.
 2. The method of claim 1,wherein said at least one mutation and/or deletion is a deletion of theregion corresponding to amino acid coordinates 99 to 131 of SEQ ID NO:8.
 3. A method of increasing the nutritional value, and/or sulfuremissions of a plant, the method comprising expressing within at least aportion of the plant an exogenous polypeptide having a cystathionineγ-synthase catalytic activity said polypeptide having an amino acidsequence corresponding to SEQ ID NO: 8 and further having at least amutation and/or deletion in a region corresponding to amino acidcoordinates 99 to 131 of SEQ ID NO: 8, thereby increasing the level ofmethionine and/or metabolites of selected from the group consisting ofS-methyl methionine, ethylene, dimethylsulfide, carbon disulfide orbiotin, hence increasing the nutritional value, and/or sulfur emissionsof the plant as compared to the nutritional value, and/or sulfuremissions of a similar wild type plant.
 4. The method of claim 3,wherein said at least one mutation and/or deletion is a deletion of theregion corresponding to amino acid coordinates 99 to 131 of SEQ ID NO:8.
 5. The method of claim 3, wherein the plant is a flowering plant, andwhereas increasing the level of methionine increases sulfur emissionsinto the scent of a flower of said flowering plant.
 6. The method ofclaim 3, wherein the plant is a consumable crop plant.